For the past several decades, the field of microbial pathogenesis has focused almost exclusively on understanding the roles of virulence factors in colonization and disease. The physiology of pathogenic bacteria has been relatively unexplored. The proposed research focuses on understanding interconnections between energy metabolism and virulence using Bordetella species as model organisms. The Bordetella genus includes several closely related subspecies of aerobic Gram-negative bacteria that colonize ciliated respiratory epithelial surfaces in mammals. In addition to their importance as infectious agents, members of the Bordetella genus provide excellent models to study bacterial-host interactions. While B. pertussis is exclusively adapted to humans, B. bronchiseptica infects a variety of laboratory animals allowing the assessment of molecular pathogen biology in a natural host-parasite context. Previous biochemical analysis by another laboratory established that virulent and avirulent Bordetella strains contain different types of cytochrome oxidases. Cytochrome oxidases are the key enzymes in aerobic respiration and directly couple to the generation of cellular energy. The differential display of these enzymes suggests that cytochrome oxidase synthesis is regulated, however, the implication for virulence is unknown. The objective of this proposal is to systematically investigate the relative contributions of cytochrome oxidases to the infectious cycle. In Aim 1, a comprehensive expression profile of all cytochrome oxidase genes will be conducted to characterize virulence-dependent and -independent mechanisms of regulation. In Aim 2, deletion and ectopic expression mutants of the cytochrome oxidase genes will be generated and characterized. The relative contributions of cytochrome oxidase to the infectious cycle will be analyzed in Aim 3 using high-resolution animal models of respiratory tract infection. The proposed work will be performed with Dr. Jeffrey F. Miller as co-investigator. Although this study is exploratory in nature Dr. Miller brings extensive expertise on Bordetella genetics and pathogen-animal host interaction to the project. [unreadable] [unreadable]